Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of this application, preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. Some material incorporated into this application has previously been published by the inventors (76).
Caffeic acid phenethyl ester, hereinafter CAPE, (FIG. 1), an agent originally isolated from propolis, a product of honeybee hives, is selectively toxic to transformed cells but not to normal cells (1). More recently, CAPE was found to inhibit the transformation mediated by adenovirus type 5 E1A, as well as the expression of the transformed phenotype in Fischer cloned rat embryo fibroblasts (2). CAPE also was more cytotoxic to transformed Fischer cloned rat embryo fibroblasts than to wild type cells. The growth of other cell lines (NIH 10T1/2, Ltk.sup.- and rat 6 cells) transformed with T24 oncogene was inhibited by CAPE, but not that of the untransformed rat 6 cells (1,2). Propolis has been considered and used in folk medicine as an anti-inflammatory agent with antitumor activity (1). One of the earlier identified propolis components that possessed anti-inflammatory and bacteriocidal properties was caffeic acid, but CAPE, which is its phenethyl ester, is more effective. The greater activity of the ester is perhaps due to its ability to more easily pass through the cell membranes. It is not known whether the free acid or its ester is the one that is active in vivo.
Reactive oxygen species (ROS), such as superoxide, H.sub.2 O.sub.2, and hydroxyl radicals, can be generated during aerobic cellular metabolism (3). In addition to their significant contribution to mutagenesis, carcinogenesis, and tumor promotion (3, 4), reactive oxygen species have been implicated in the etiology and pathophysiology of many human disease, including rheumatoid arthritis, systemic lupus erythematosus, sickle cell anemia, and various forms of cancer (3, 5, 6). Reactive oxygen species induce strand breaks in DNA and oxidative modification of DNA bases, which are implicated in the mutagenic and carcinogenic effects of reactive oxygen species (3, 7-9). Although the oxidized bases can be repaired by DNA glycosylases and/or endonucleases (10-15), when the repair is not complete or timely, deleterious effects may take place. 5-hydroxymethyl-2'-deoxyuridine (HMdUrd) is cytotoxic and cytostatic to a number of mammalian cells and is mutagenic, while 8-hydroxyl-2'-deoxyguanosine (8-OHdGua) can serve as a mispairing lesion during cellular DNA replication (9, 15-17). 8-OHdGua has been widely used as an important biological marker for carcinogenesis and cellular oxidative stress (3, 18, 19).
It has recently been shown that the phorbol ester-type tumor promoters (12-O-tetradecanoylphorbol-13-acetate, hereinafter TPA) induce H.sub.2 O.sub.2 production in mouse skin as well as cause oxidation of DNA bases in vivo (20-22). In addition, it has been found that agents possessing anti-tumor-promoting properties in vivo, also suppress inflammatory processes. Processes suppressed by such agents include infiltration of polymorphonuclear leukocytes (hereinafter PMNs), reactive oxygen species production, and oxidation of DNA bases (20-22), as well as induction of ornithine decarboxylase (ODC) and edema (23-26). A number of known anti-tumor promoters that possess all or some of those properties have been isolated from biological sources, and include sarcophytol A (isolate from marine soft coral) (27, 28), (-)-epigallocatechin gallate (EGCG, a polyphenol from green tea) (26, 29, 30), curcumin (a spice) (24, 25), and caffeic acid (24, 25).
TPA has been found to induce oxidative stress in bovine eye lens and also causes its opacity (31). Some of the chemopreventive agents (EGCG and sarcophytol A) used in tumor promotion studies were also effective inhibitors of TPA-mediated lens opacification and H.sub.2 O.sub.2 production (31).
H.sub.2 O.sub.2 and oxygen free radicals participate in cellular aging in humans. Cataract formation in humans is primarily associated with advanced age (32, 33, 34). Indeed, cataract development is one disease thought to be triggered by this kind of oxidative stress (34, 35, 39). In fully developed cataracts in humans and in several experimental cataract models, the oxidation of proteins (40, 41) and the peroxidation of lipids in lens tissue appears to be initiated by reactive oxygen species (33, 41-43).
As life expectancy increases, the burden of cataract formation in humans in terms of suffering and cost increases. Worldwide, cataracts are the leading cause of blindness. About 50% of individuals in the United States over 65 years of age have some stage of cataract development and about 1.3 million surgical cataract procedures are performed annually (Paton and Craig, 1990). At present, there is no proven non-surgical modality to cure cataracts, nor to retard the development of any form of cataract regardless of age.
Since the mechanism of the action of CAPE is not known, we set out to establish whether it possesses some of the properties that are common to a number of chemopreventive agents (3). Such properties include inhibition of ROS production and oxidative damage to cellular macromolecules, as well as edema and ornithine decarboxylase (ODC) induction, which are thought to contribute to tumor promotion and/or progression (3, 20-30, 44-47). Furthermore, we decided to establish whether CAPE prevents TPA-mediated ROS generation by the lens, as well as lens opacity, properties that would be useful in the prevention of cataracts (39). We tested the therapeutic use of CAPE to mitigate and delay the progression of cataracts in intact animals.
The human immunodeficiency virus enzyme HIV integrase mediates the integration of the HIV DNA into the genome of a host. It has been shown that CAPE inhibits HIV integrase (48). By combining CAPE with a substance which inhibits an HIV enzyme other than HIV integrase, HIV may be blocked at two different replication stages. We disclose herein a pharmaceutical composition comprising CAPE and a substance which inhibits HIV replication, the pharmaceutical composition being useful for treating HIV infections.